Preparation of competent cells
Growth of bacterial cells
- Innoculate 80 mL LB in a 500 mL flask with three 2-3 mm colonies from fresh plates (BL21 or DH5α)
- Incubate at 37 °C with shaking (220-300 rpm) until A600= 0.4
- For optimal transformation efficiency, it is essential for the cells to be in log growth phase. For this reason, be sure to continue to the next step of the protocol before the A600 goes above 0.6
Competent cell treatment
- Transfer the culture to sterile 50 mL tubes and chill on ice for 15 minutes.
- Harvest the cells by centrifugation at 1,000 g for 15 minutes at 4 °C.
- Decant the supernatant and carefully remove all remaining LB.
- Add RFI to 1/3 the original culture volume (~2.5 mL)
| Buffer RFI Components|| Buffer RFI Recipe (100 mL)
10.2 mM CaCl2
30 mM KOAc
12.5 g RbCl
0.99 g MnCl2•H2O
3 mL of 1 M KOAc pH 7.5
15 mL glycerol
H2O to 90 mL
- Carefully adjust pH to 5.8 with 0.2 M acetic acid if necessary. If pH drops below 5.78, salts will precipitate and the solution will be unusable
- Sterilize by 0.22 μm filter (prerinsed)
Aliquoting & storage
- Dispense 100 μL aliquots into labeled, pre-cooled 0.5 mL tubes.
- Flash-freeze with liquid N2.
- When fully-frozen, store at -80 °C.
Adapted from "Preparation of competent cells" by Jonathan Ipsaro, Mondragon Lab, Northwestern University, USA